Flotillin-1 regulates oncogenic signaling in neuroblastoma...

1

Flotillin-1 regulates oncogenic signaling in neuroblastoma cells by regulating

ALK membrane association

Arata Tomiyama1,2

, Takamasa Uekita1,3

, Reiko Kamata1, Kazuki Sasaki

4, Junko

Takita5, Miki Ohira

6, Akira Nakagawara

7, Chifumi Kitanaka

8, Kentaro Mori

2,

Hideki Yamaguchi1, and Ryuichi Sakai

1

1) Division of Metastasis and Invasion Signaling, National Cancer Center Research

Institute, Tokyo, Japan.

2) Department of Neurosurgery, National Defense Medical College, Saitama, Japan.

3) Department of Applied Chemistry, National Defense Academy, Kanagawa, Japan

4) Department of Molecular Pharmacology, National Cerebral and Cardiovascular

Center Research Institute, Osaka, Japan.

5) Department of Cell Therapy and Transplantation Medicine, Graduate School of

medicine, The University of Tokyo, Tokyo, Japan.

6) Division of Cancer Genomics, Chiba Cancer Center Research Institute, Chiba,

Japan.

7) Division of Biochemistry and Innovative Cancer Therapeutics, Chiba Cancer Center

Research Institute, Chiba, Japan.

8) Department of Molecular Cancer Science, Yamagata University School of Medicine,

Yamagata, Japan.

[Running title]

Flotillin-1 regulates ALK signaling in neuroblastoma

[Key words]

Anaplastic lymphoma kinase, Flotillin-1, Neuroblastoma, Endocytosis, Oncogenicity

[Corresponding Author]

Ryuichi Sakai

Division of Metastasis and Invasion Signaling, National Cancer Center Research

Institute

5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan

(Phone): +81-3-3542-2511

Research. on May 11, 2018. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 15, 2014; DOI: 10.1158/0008-5472.CAN-14-0241

http://cancerres.aacrjournals.org/

2

(Fax): +81-3-3542-8170

(E-mail): [email protected]

[Disclosure of Potential Conflicts of Interest]

No potential conflicts of interests were disclosed.

Total number of figures: 6

Total number of supplementary figures: 8

Word count: 4967



mailto:[email protected]


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Abstract

Neuroblastomas harbor mutations in the non-receptor kinase ALK in 8-9% of cases

where they serve as oncogenic drivers. Strategies to reduce ALK activity offer clinical

interest based on initial findings with ALK kinase inhibitors. In this study, we

characterized phosphotyrosine-containing proteins associated with ALK to gain

mechanistic insights in this setting. Flotillin-1 (FLOT1), a plasma membrane protein

involved in endocytosis, was identified as a binding partner of ALK. RNAi-mediated

attenuation of FLOT1 expression in neuroblastoma cells caused ALK dissociation

from endosomes along with membrane accumulation of ALK, thereby triggering

activation of ALK and downstream effector signals. These features enhanced the

malignant properties of neuroblastoma cells in vitro and in vivo. Conversely,

oncogenic ALK mutants showed less binding affinity to FLOT1 than wild-type ALK.

Clinically, lower expression levels of FLOT1 were documented in highly malignant

subgroups of human neuroblastoma specimens. Taken together, our findings suggest

that attenuation of FLOT1-ALK binding drives malignant phenotypes of

neuroblastoma by activating ALK signaling.

(158 words)




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Introduction

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that

is rather specifically expressed in the nervous system during development in mice (1).

ALK was first identified in anaplastic large cell lymphoma as the fusion protein

NPM-ALK caused by chromosomal translocation (2). Recently, ALK was highlighted

as a therapeutic target of several cancers such as non-small-cell lung cancers and

colon cancers, which possess oncogenic fusion ALK proteins such as EML4-ALK (3-

6). Genetic alterations of ALK have also been identified in cell lines and clinical

samples of neuroblastoma, which consist of gene amplifications, activating mutations,

or N-terminus truncations (7-12). Activated ALK proteins in neuroblastoma are

distinct from other tumors as for the point that they retain the transmembrane domain.

The survival of neuroblastoma cells with activated ALK is dependent on the ALK

protein in some cases, which highlights the so called oncogene addiction to activated

ALK (13).

Neuroblastoma is one of the most refractory solid tumors in children with 5-

year survival rates of less than 40% following conventional treatments (14-16). To

this end, clinical trials involving neuroblastoma patients and ALK inhibitors such as

crizotinib have already begun (17). However, it was reported that neuroblastoma

harboring certain types of activation mutations of ALK show greater resistance to the

ALK inhibitors (18) and that there are differences in the malignancy grades among

neuroblastoma cases with mutant ALK depending on the type of mutations (19, 20).

Therefore, further investigation is necessary to elucidate what aspects of the mutant

ALK protein determine the clinicopathological features of neuroblastoma.

As ALK is a receptor tyrosine kinase, it is essential to understand the signal

transduction pathways which mediate the activation of this kinase. In addition to the

common downstream mediators of receptor tyrosine kinases, such as Akt, Erk, and

STAT3, we have shown the critical role of ShcC as a binding partner of ALK in

neuroblastoma (21, 22). Further identification of the tyrosine-phosphorylated binding

partners of ALK and analysis of their functions in neuroblastoma will aid

understanding of the unique oncogenic roles of ALK signaling.

FLOT1 is a plasma membrane lipid raft-localizing protein that is involved in

internalization of membrane-localizing proteins into the cytosol by endocytosis. In

addition, FLOT1 plays a role in the regulation of actin organization and neuronal




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regeneration (23, 24) and phosphorylation of FLOT1 at the tyrosine or serine is

necessary during internalization (25, 26). At present, there is only limited information

regarding the involvement of FLOT1 in the oncogenicity of solid cancers other than

neuroblastoma (27-29). In this report, we identified FLOT1 during the screening of

ALK-binding tyrosine-phosphorylated proteins in neuroblastoma cells by using mass

spectrometry analysis. Functional analysis revealed that FLOT1 controls the

malignant properties of neuroblastoma by regulating the endocytosis and degradation

of membrane-localizing ALK protein. It was also suggested that alterations to the

binding affinity to FLOT1 in some of the ALK mutants might contribute to the

enhancement of oncogenic ALK signaling in neuroblastoma.




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Materials and Methods

Antibodies and plasmids

The rabbit ALK antibody was previously described (22). The antibodies

against phospho-ALK, Akt, phospho-Akt, p44/42 MAPK (ERK1/2), phospho-

ERK1/2, STAT3, phospho-STAT3, and p53 were purchased from Cell Signaling

Technology. Other antibodies used are: ALK (H260), clathrin HC, and LAMP2 (Santa

Cruz Biotechnology); FLOT1, N-cadherin, and caveolin-1 (BD Transduction

Laboratories); FLAG M2 and -tubulin (Sigma); HA (Nakarai Tesque); and

phosphotyrosine (4G10; Upstate Biotechnology).

The cDNAs of human wild-type ALK, the activating mutants of ALK (F1174L,

K1062M, and R1275Q), and wild-type FLOT1 were subcloned into the pcDNA3.1

vector.

Cell culture and tissue samples

NB-39-nu and Nagai human neuroblastoma cell lines were provided by

Carcinogenesis Division, National Cancer Center Research Institute in 2001 (30).

TNB-1 human neuroblastoma cell line was obtained from Human Science Research

Resource Bank in 2001 (31). Gene amplification of MYCN in these 3 lines and of ALK

in NB-39-nu and Nagai is periodically checked to confirm the neuroblastoma origin

of these cell lines, most recently in March 2014 (22). The cells were maintained in

RPMI 1640 medium (Invitrogen) supplemented with 10% FBS, 10 units/mL penicillin,

and 10 µg/mL streptomycin at 37 °C in a humidified atmosphere containing 5 % CO2.

Human neuroblastoma tissue samples were prepared as previously described (32).

Transfection and establishment of stable clones

20 nM of Stealth Select RNAi (Invitrogen) or 4 µg of plasmid was transfected

by electroporation using the NEON system (Invitrogen). The siRNA sequences are

described in Supplementary Materials and Methods. For establishment of stable ALK

mutant clones, TNB-1 cells were continuously treated with 400 µg/mL of G418.

TNB-1 cells stably expressing control or FLOT1 shRNA were established using

lentiviral particles according to the manufacturer’s instructions (Santa Cruz

Biotechnology).




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Purification of ALK-binding tyrosine-phosphorylated proteins

The immunoafffinity purification methods previously described (33) were

modified and used for isolation of the ALK-binding tyrosine-phosphorylated proteins.

The detailed protocol is described in Supplementary Materials and Methods.

Immunoblotting, immunoprecipitation, and immunofluorescence

The immunoblotting, immunoprecipitation, and immunofluorescence were

done as described previously (26, 32) with modifications. The detailed protocols are

described in Supplementary Materials and Methods.

Pulse-chase analysis of ALK internalization

Cells cultured on coverslips were incubated with cold complete medium for 5

minutes at 4 °C and then with medium containing 4 µg/mL of anti-ALK (H260)

antibody for 30 minutes at 4 °C. After removing the medium, the cells were cultured

in fresh medium at 37 °C for the indicated time period. The cells were fixed and

stained with the fluorescence-conjugated secondary antibody. For colocalization

analysis, the cells were also stained for FLOT1, clathrin, or caveolin-1. The cells have

cytosolic colocalization signals (diameter > 2 µm) were counted using fluorescence

images and Image J software. At least 200 cells per sample were counted, and the

percentage of positive cells was calculated.

Biotinylation and purification of plasma membrane-localized proteins

5 x 107 cells were incubated with cold complete medium for 5 minutes at 4 °C.

The cell surface proteins were labeled with 200 g sulfo-NHSS-biotin (Thermo

Scientific) for 40 minutes at 4 °C. After cell lysis, biotinylated proteins were

immunoprecipitated using Ultralink Immobilized NeutrAvidinTM

protein (Thermo

Scientific). For internalization assay, the labeled cells were cultured in fresh complete

medium at 37 °C for 60 minutes. The cell surface biotin was stripped by incubation

with 180 mM sodium 2-mercaptoethane sulfonate (MesNa; Sigma). After quenching

the MesNa by the addition of 180 mM iodoacetamide (Sigma) for 10 minutes, the

biotinylated proteins were immunoprecipitated.

Cell migration assay

The cells (1 × 104) were seeded onto the upper part of the transwell inserts




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(BD Falcon) coated with fibronectin. The migrated cells on the lower surface of the

filter were fixed and stained with Giemsa’s stain solution. The number of migrated

cells was counted using a BX51 microscope (Olympus).

Cell death assay

The cellular nuclei stained with Hoechst 33342 and PI (Thermo Scientific)

were independently counted using a fluorescence microscope (IX81-ZDC-DSU;

Olympus). At least 500 cells per sample were examined and the percentage of PI-

positive cells to total Hoechst-positive cells was calculated.

Anchorage-independent cell proliferation assay

Cells were cultured on MPC-coated plates (Thermo Scientific) at 1 × 103 cells

per six wells for 7 days and the total numbers of cells were counted.

Tumor xenograft assay

The animal experimental protocols were approved by the Committee for

Ethics of Animal Experimentation, and the experiments were conducted in accordance

with the guidelines for animal experiments in the National Cancer Center. TNB-1

cells (5 × 106) were subcutaneously injected into the bilateral flank of female 6-week-

old BALB/c nude mice (Clea Japan). At 6 weeks after tumor inoculation, the mice

were sacrificed, and the subcutaneous tumors were excised with the attached muscle

layers. The tumor volume was calculated with the equation (length × width2)/2 and

the tumor weight (g) was measured. The tumor tissue was stained by H&E.

Statistical analysis

The data for all the quantitative results are expressed as mean and standard

deviation from three independent experiments. Plotting of scatter graphs and testing

of difference of means by Student’s t-test were achieved using Microsoft Excel 2007

software. P-values of <0.01 were considered as statistically significant.




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Results

Identification of FLOT1 as a binding partner and kinase substrate of ALK in

neuroblastoma

To identify the phosphotyrosine-containing proteins associated with anaplastic

lymphoma kinase (ALK), we performed two-step affinity purification using TNB-1

neuroblastoma cells, which stably expresses the ALK protein tagged with FLAG at

the C-terminus as described in Supplementary Fig. S1. Mass spectrometry analysis

identified several reported binding partners of ALK such as ShcA, ShcC and IRS1 (22,

34) along with numbers of novel candidates of ALK-binding phosphoproteins.

Association of these novel candidates with ALK was confirmed by

immunoprecipitation analysis using available antibodies and further association with

prognosis of neuroblastoma was checked using public database in order to estimate

clinical impact. In this study, we focused on flotillin-1 (FLOT1) among these ALK-

binding proteins through these screening.

The R2 database, one of the largest public databases of microarray in

neuroblastoma cases (http://r2.amc.nl), indicated that high expression levels of ALK

mRNA significantly correlates with poor prognosis in neuroblastoma patients (Fig.

1A) suggesting that ALK signaling has clinical impact even in patients without

genetic alteration of ALK. On the other hand, low expression of FLOT1 mRNA was

positively correlated with poor prognosis of clinical neuroblastoma cases in the R2

database (Fig. 1B). We also analyzed the expression of FLOT1 and ALK proteins in

specimens from 45 clinical neuroblastoma cases, which belong to three clinical

malignancy grades (favorable, 15 cases; intermediate, 18 cases; unfavorable, 12

cases) as classified by Brodeur’s classification (35, 36), and demonstrated that the

levels of FLOT1 expression inversely correlate with clinical malignancy grade (Fig.

1C). A representative blot of 5 samples from each group is shown in Supplementary

Fig. S2.

Because FLOT1 expression has apparent association with prognosis and

clinical grades of neuroblastoma, we hypothesized that FLOT1 regulates oncogenic

potentials of neuroblastoma through association of ALK. The binding of ALK to

FLOT1 was confirmed by immunoprecipitation analysis using anti-FLAG or anti-HA

antibodies in COS-7 cells expressing FLAG-tagged ALK and HA-tagged FLOT1 (Fig.

2A). Binding of ALK to endogenous FLOT1, as well as ALK-mediated tyrosine-




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phosphorylation of FLOT1, was also demonstrated in NB-39-nu neuroblastoma cells

harboring amplified ALK (Fig. 2B and Supplementary Fig S3). By

immunocytostaining analysis, ALK and FLOT1 were mainly co-localized within the

cytoplasm, especially at the submembrane regions of the ventral membrane (Fig. 2C).

These results suggested that FLOT1 is associated with ALK as a binding partner and

kinase substrate in neuroblastoma cells.

FLOT1 regulates degradation of ALK in lysosome through endocytosis

Considering that FLOT1 is reported to be involved in endocytosis of

membrane proteins, we investigated the effect of FLOT1 knockdown on the amount

of membrane-localizing ALK. The amount of ALK protein at the plasma membrane

was markedly increased by treatment with either of two FLOT1 siRNAs, which

resulted in rather moderate increases in total ALK protein levels (Fig. 3A). Pulse-

chase analysis with an ALK antibody revealed marked reduction in the amount of

internalized ALK in the NB-39-nu cells treated with each FLOT1 siRNA at the time

point of 30 minutes (Fig. 3B). Biotinylation-internalization analysis confirmed that

gradual increase in the total amount of internalized ALK was significantly impaired

by treatment with each FLOT1 siRNA (Fig. 3C). These results suggested that FLOT1

regulates the amount of ALK on the cell surface through endocytosis.

Membrane proteins that are internalized by endocytosis are usually degraded

by the proteasome or lysosome (37). Degradation of ALK was inhibited following

treatment with the lysosomal inhibitor concanamycin, while it was not significantly

affected by the proteasomal inhibitor MG132 (Fig. 3D). Under the presence of

concanamycin, accumulation of ALK at plasma membrane was observed by

knockdown of FLOT1 whereas total ALK protein level was less affected

(supplementary Fig. S4A). Pulse-chase analysis visualized by immunocytostaining

demonstrated the colocalization of internalized ALK with the lysosomal marker

LAMP2 that was disrupted by treatment with FLOT1 siRNA (Fig. 3E).

Colocalization of FLOT1 with internalized ALK was also observed at the early phase

of endocytosis, while no obvious colocalization of ALK with the other known

endosomal transporters, clathrin heavy chain and caveolin-1 (38, 39), was observed

(Supplementary Fig. S4B and C). These results indicated that FLOT1 regulates

lysosomal degradation of ALK through clathrin/caveolin-independent endocytosis.




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FLOT1 regulates ALK signaling through modulation of the amount of cell-

surface ALK

Phosphorylation of ALK as well as known downstream mediators of ALK

such as AKT, ERK1/2 and STAT3, was increased in the NB-39-nu cells treated with

FLOT1 siRNA (Fig. 4A). The increased levels of phosphorylation of these molecules

were all subsequently reduced by treatment with either ALK siRNA or NVP-TAE-684,

an inhibitor of ALK. We further analyzed whether the expression of FLOT1 affects

the oncogenic properties of activated ALK in NB-39-nu neuroblastoma (13, 22).

Induction of anchorage independent growth, resistance to the anticancer agent cis-

diamminedichloroplatinum (cisplatin; CDDP), and cell migration were enhanced by

the treatment of NB-39-nu cells with FLOT1 siRNA (Fig. 4B, Supplementary Fig.

S5A and B). Similar results were also obtained using Nagai, another neuroblastoma

cell line harboring amplified wild-type ALK (Supplementary Fig. S6A and B). On the

other hand, reduced expression and phosphorylation of ALK, and phosphorylation of

AKT, ERK1/2, and STAT3 as well as induction of cell death, decreased proliferation,

and acceleration of ALK internalization were observed by overexpression of FLOT1

in NB-39-nu cells (Fig. 4C, 4D, Supplementary Fig. S5C, S5D).

To investigate whether FLOT1 has the same regulatory roles of ALK in

neuroblastoma cells harboring single-copy ALK, TNB-1 cell lines stably expresses

FLOT1 shRNA, TNB-FL1 and TNB-FL2 were established (Fig. 4E). Two control

lines of TNB-1 cells, TNB-Con1 and TNB-Con2 cells, were also established using the

control vector. Enhanced expression of ALK and phosphorylation of AKT and

ERK1/2 was observed in TNB-FL1 and TNB-FL2 cells, while no significant changes

in the expression of other receptor tyrosine kinases (RTKs), such as EGFR, RET, and

TrkB were observed (Fig. 4E). In addition, increased anchorage-independent growth

was detected in the TNB-FL1 and TNB-FL2 cells, which was blocked by treatment

with the ALK inhibitor (Fig. 4F and Supplementary Fig. S5E). These results

demonstrated that FLOT1 inhibits the malignant phenotype of neuroblastoma cells

through endocytosis of ALK.

Activating mutations of ALK have low binding affinities to FLOT-1 and cause

ALK stabilization and malignant phenotypes in neuroblastoma cells

It is reported that some of the activating mutations of ALK such as the

common F1174L mutation exhibit more malignant phenotypes and poor prognosis




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than others (19) while the mechanisms causing the differences are still not clear. We

investigated differences in the binding affinities between mutant ALK proteins and

FLOT1 by using TNB-1 neuroblastoma cells stably expressing wild-type (WT),

F1174L (FL; mutation near the c-helix loop), K1268M (KM; mutation in the

juxtamembrane domain), and R1275Q (RQ; mutation near the ATP-binding domain)

mutants of ALK (8-11). FLOT1 steadily associated with the WT and RQ mutants of

ALK, but not as efficiently with the FL and KM mutants (Fig. 5A). Furthermore,

knockdown of FLOT1 affected the internalization of biotinylated ALK in the WT and

RQ mutants but not in the FL and KM mutants (Fig. 5B). In addition, the

phosphorylation levels of ALK, AKT, and ERK1/2 were not obviously elevated by

treatment with FLOT1 siRNA in the TNB-1 cells with the FL or KM mutation (Fig.

5B).

Anchorage-independent growth was significantly enhanced by FLOT1

siRNA in cells expressing WT or RQ mutant, whereas no obvious changes were

observed in the cells expressing the FL and KM mutants, which originally showed

enhanced anchorage-independent growth. Anchorage-independent growth of all the

cells analyzed was reduced by treatment with the ALK inhibitor (Fig. 5C). Similar

difference in ALK mutants were also confirmed using the TNB-1 cells expressing WT

ALK, the FL mutant, and the KM mutant as for resistance to CDDP and cell

migration, (Supplementary Fig. S7A and B). These results suggested that some of the

activating mutations of ALK might have enhanced stability at the cell membrane by

reduced affinity to FLOT1, which leads to further enhancement of the malignancy of

neuroblastoma.

FLOT1 regulates tumorigenicity of neuroblastoma cells

To investigate the role of FLOT1 in the tumorigenicity of neuroblastoma,

TNB-Con1/2 and TNB-FL1/2 cells were subcutaneously injected into nude mice (Fig.

6A). Because of the low tumorigenicity of the original TNB-1 cells, tumors were not

detectably formed at 6 weeks following injection of TNB-Con cells. On the other

hand, tumors as large as 10–40 mm in diameter were clearly formed by the TNB-FL1

and TNB-FL2 cells in this period (Fig. 6A). Histological study of these tumors

revealed that the tumor cells had infiltrated into the muscle layers and formed large

intratumoral vessels, which reflects the malignant phenotype of the tumors (Fig. 6B).

These results indicated that FLOT1 might be a negative regulator of the malignant




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characteristics of neuroblastoma in vivo. Along with the results indicating the low

expression of FLOT1 is significantly associated with poor prognosis and unfavorable

histological grades of neuroblastoma (Fig. 1B & 1C), it was indicated deregulation of

FLOT1 expression is involved in the progression of neuroblastoma through

enhancement of ALK signaling.




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Discussion

Deregulation of the receptor tyrosine kinase ALK by amplification or

activating mutation of the ALK gene has been reported in 10%–15% of human

neuroblastoma cases, in which the relationship between ALK signaling and

oncogenesis of neuroblastoma is indicated. While the association between ALK

expression and poor prognosis of neuroblastoma is observed (Fig.1A), it is not clear

whether different modes of activation of ALK signaling are involved in the

progression of the other neuroblastoma cases. In this study, we provided in vitro and

in vivo evidence that activation of ALK signaling caused by impaired FLOT1-

mediated endocytosis is associated with malignancy of neuroblastoma cells. This

finding was supported by the observation that FLOT1 expression levels in the clinical

samples is inversely correlated with prognosis of the disease in a public database (Fig.

1B) and grades of malignancy in tissue samples (Fig. 1C). Taken together, the novel

tumor-suppressing role of FLOT1 in the majority of neuroblastoma that lacks genetic

alterations of ALK was implied.

There was tendency that the low expression level of FLOT1 is associated

with high expression levels of ALK in the neuroblastoma tissues used for Fig.1C,

while it was not statistically significant possibly due to limited numbers of tissues

examined (data not shown). Therefore, we could not completely exclude the

possibility that FLOT1 also degrades signaling molecules other than ALK, which are

associated with malignancy of neuroblastoma, although it was confirmed that FLOT1

preferably regulates the ALK protein among several other RTKs expressed in

neuroblastoma (Fig. 4E). The information regarding the involvement of FLOT1 in

cancer development is still limited (29, 40-42). It was recently suggested that FLOT1

is associated with poor prognosis of breast cancer as a result of stabilization of ErbB2

(27) and also plays oncogenic roles in esophageal cancer and hepatocellular

carcinoma (28, 40). It is speculated that FLOT1 might regulate the organ-dependent

target proteins and functions in the development of cancers and the regulation is

rather selective to ALK in neuroblastoma. Considering that activated ALK found in

other types of cancers lack transmembrane domain, the accumulation of membranous

ALK by deficient FLOT1 might be etiological only in neuroblastoma.

It has been reported that FLOT1 physiologically acts as an endosomal

transporter of membrane proteins (23-25). Further study is required to clarify the




15

precise mechanisms of endocytosis of ALK including mode of association between

FLOT1 and ALK and the role of tyrosine phosphorylation of FLOT1 in this process.

It has been reported that another RTK-binding protein Cbl is involved in the

downregulation of RTKs through receptor ubiquitination followed by endocytosis.

Indeed, some mutations in the RTKs Met and EGFR decrease the affinity of RTKs

with Cbl, which results in impaired endocytosis and oncogenic accumulation of RTKs

in several cancers including lung cancer and glioblastoma (43).

It was recently reported that neuroblastoma cases harboring certain

activation-mutations of ALK exhibit higher refractoriness (19). For example,

neuroblastoma cases harboring the F1174L mutant are also reported to have higher

resistance than cases with amplified or R1275Q mutant ALK when treated with an

ALK inhibitor (18). Both the F1174L and R1275Q mutations of ALK are frequently

observed, while F1174L mutant is more aggressive and resistant to ALK inhibitors

than R1275Q mutants even in a transgenic fly system (20). We demonstrated that the

FL and KM mutants of ALK has less affinity to FLOT1 and therefore less affected by

FLOT1-mediated endocytosis than the wild type ALK (Fig. 5B). The role of

oncogenic potential of ALK mutants has previously been analyzed with respect to

ATP-binding potential and impaired receptor trafficking (44, 45), while our results

suggest that impaired downregulation of ALK by FLOT1 might contribute to the

aggressiveness of some mutations of ALK (Supplementary Fig. S8). It should be

emphasized that expression levels of FLOT1 might also become one of the efficient

clinical markers determining prognosis and therapeutic effectiveness of ALK

inhibitors in neuroblastoma cases.




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Grant Support

This work was supported by the National Cancer Center Research and Development

Fund (25-B-3) and by Grants-in-Aid for Scientific Research (B) by the Ministry of

Education, Culture, Sports, Science and Technology of Japan.




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Figure legends

Figure 1. Clinical impact of FLOT1 expression in neuroblastoma cases

(A and B) Kaplan-Meier analysis of overall survival in patients with neuroblastoma

with the classifications based on anaplastic lymphoma kinase (ALK) (A) and

flotillin-1 (FLOT1) (B) mRNA expression. The data were obtained from the R2

microarray public database (http://r2.amc.nl).

(C) The protein samples from clinical neuroblastoma specimens classified by

Brodeur’s classification (Favorable, 15 cases; Intermediate, 18 cases;

Unfavorable, 12 cases) were subjected to immunoblotting using the FLOT1

antibody and the expression levels of FLOT1 were quantified. Red bar denote the

average values. *P < 0.01

Figure 2. FLOT1 interacts with ALK in neuroblastoma cells

(A) Cos-7 cells were transiently transfected with an empty vector (vector), flag-

tagged wild-type ALK, or HA-tagged wild-type FLOT1 for 12 hours. The cell

lysates were immunoprecipitated with control IgG, anti-FLAG antibody (ALK),

or anti-HA antibody (FLOT1) and the immunocomplexes and total cell lysates

(Input) were analyzed by immunoblotting using the indicated antibodies.

(B) NB-39-nu neuroblastoma cells were transiently transfected with control siRNA

(Con) or one of two siRNAs against ALK (ALK1 and ALK2) for 72 hours. The

cell lysates were immunoprecipitated with the indicated antibodies and subjected

to immunoblotting.

(C) NB-39-nu cells were stained with DAPI (blue) and antibodies against ALK (red)

and FLOT1 (green), and observed by confocal microscopy. The submembrane

regions of the dorsal cell membrane were imaged. Lower panels are magnified

images of the boxed regions. Arrows indicate co-localization of ALK and FLOT1.




22

Bar, 10 µm.

Figure 3. FLOT1 regulates endocytosis and cell surface expression of ALK

(A) NB-39-nu cells were treated with control siRNA (con) or FLOT1 siRNA (FL1

and FL2) for 72 hours. The plasma membrane-localized proteins were purified as

described in the Materials and Methods and analyzed by immunoblotting using

the ALK antibody or N-cadherin antibody. Total cell lysates were also analyzed by

immunoblotting using the indicated antibodies. The levels of ALK in each sample

were quantified and denoted as relative value (siRNA(-)=1) under immunoblotting

data.

(B) NB-39-nu cells treated with indicated siRNAs were subjected to pulse-chase

analyses by using an ALK antibody (red). The cells were stained with DAPI (blue)

and FLOT1 antibody (green) and observed by confocal microscopy. The lower

images are magnified images of the boxed regions. Fluorescent intensity of each

signals were quantified by line scan analysis as indicated by yellow arrows and

depicted as histograms. Open arrows and circles indicate peaks of the fluorescence

signals for the plasma membrane and the cytosol, respectively. Bar, 10 µm.

(C) The siRNA-treated NB-39-nu cells were subjected to ALK-internalization assays

at the indicated time points as described in the Materials and Methods. The avidin-

bounded (internalized) proteins and total cell lysates were analyzed by

immunoblotting using the indicated antibodies.

(D) NB-39-nu cells treated with siRNAs were cultured in the presence of DMSO,

lysosomal inhibitor concanamycin (Cc, 10 µM), or proteasomal inhibitor MG132

(MG, 15 µM) for 8 hours and subjected to immunoblotting. p53 was analyzed as a

representative protein degraded by proteasome.

(E) NB-39-nu cells were treated with siRNAs and pulse-chased with an ALK

antibody (red). The cells were stained with DAPI (blue) and LAMP2 antibdy




23

(green). Lower panels are magnified images of the boxed regions. Cells with

ALK-positive and LAMP2-positive dots were quantified as described in the

Materials and Methods (right bar graph). Bar, 10 µm. *P < 0.01

Figure 4. FLOT1 regulates ALK signaling and oncogenicity of neuroblastoma

cells

(A) NB-39-nu cells were treated with control (Con) or FLOT1 siRNA (FL1 or FL2)

alone or in combination with ALK siRNA (ALK1 or ALK2) for 72 hours. FLOT1

siRNA-treated cells were further cultured in the presence DMSO or ALK inhibitor

NVP-TAE-684 (TAE; 20 nM) for 2 hours. The cell lysates were analyzed by

immunoblot using the indicated antibodies.

(B) NB-39-nu cells were treated with indicated siRNAs and their anchorage-

independent growth under continuous treatment with DMSO or TAE was tested as

described in the Materials and Methods.

(C) NB-39-nu cells were transiently transfected with an empty vector or HA-tagged

FLOT1 for the indicated times. The cell lysates were analyzed by immunoblot

using the indicated antibodies.

(D) NB-39-nu cells transfected with vector alone or HA-tagged FLOT1 were cultured

with DMSO or the caspase inhibitor z-VAD-FMK (Z-VAD; 100 µM) for 72 hours.

Cell proliferation was mesured as described in the Materials and Methods.

(E) Stable transfectants of TNB-1 cells expressing control shRNA (TNB-Con1 and

TNB-Con2) or FLOT1 shRNA (TNB-FL1 and TNB-FL2) were analyzed by

immunoblotting. The expression levels of receptor tyrosine kinases were

quantified and denoted as relative value (TNB-Con1=1) under each

immunoblotting data.

(F) The anchorage-independent growth of TNB-1 cells expressing control or FLOT1

shRNA was tested as described in the Materials and Methods.




24

Figure 5. Activating mutations of ALK confer resistance to downregulation by

FLOT1

(A) Cell lysates from each pair of different stable TNB-1 transfectants of empty

vector and ALK mutants (WT, wild-type; FL, F1174L; KM, K1062M; RQ,

R1275Q) were immunoprecipitated using the anti-ALK antibody. The

immunocomplexes and total cell lysates were analyzed by immunoblotting.

(B) The stable TNB-1 transfectants were transfected with control siRNA (con) or

FLOT1 siRNA (FL1 or FL2) for 48 hours. The cells were subjected to the ALK

internalization assay for 60 minutes, and the internalized proteins and total cell

lysates were analyzed by immunoblotting.

(C) The stable TNB-1 cells transfectants were transfected with indicated siRNAs for

48 hours and cultured in the presence of DMSO or ALK inhibitor NVP-TAE-684

(TAE; 20 nM) for 2 hours. The cells were subjected to the anchorage-

independent cell growth assay under continuous treatment with DMSO or TAE.

*P < 0.01

Figure 6. FLOT1 negatively regulates tumorigenicity of neuroblastoma cells

(A) (Left: ) Macroscopic images of the mouse xenograft experiment. The TNB-1 cells

stably expressing control (TNB-Con1 and -Con2, yellow circle) or FLOT1 shRNA

(TNB-FL1 and –FL2, red circle) were subcutaneously inoculated into both sides

of the flank of 4-week-old nude mice as shown (See Supplementary Materials and

Methods). In the first group (Group 1) TNB-Con1 and –FL1 cells, and in the

second group (Group 2) TNB-Con2 and –FL2 cells were inoculated. The images

of one of the five mice from each group 1 and group 2 at 6 weeks after inoculation

were presented.

(Right: ) The total tumor volume and weight were measured and plotted as

scattergrams. Green bars denote the average values. *P < 0.01




25

(B) High magnification image of the tumor tissue stained with H&E from the

mouse (Group 1, TNB-FL1 cells). Tumor invasion into the subcutaneous muscle

layer (Mus) and the formation of intratumoral large vessels (Ves) were observed.

The dotted line denotes the margin of the muscle layer. Bar, 100 µm.




Published OnlineFirst May 15, 2014.Cancer Res Arata Tomiyama, Takamasa Uekita, Reiko Kamata, et al. cells by regulating ALK membrane associationFlotillin-1 regulates oncogenic signaling in neuroblastoma

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